The role of CD4+ T cells in visceral leishmaniasis; new and emerging roles for NKG7 and TGFß.

Visceral leishmaniasis is a potentially devastating neglected tropical disease caused by the protozoan parasites Leishmania donovani and L. infantum (chagasi). These parasites reside in tissue macrophages and survive by deploying a number of mechanisms aimed at subverting the host immune response. CD4+ T cells play an important role in controlling Leishmania parasites by providing help in the form of pro-inflammatory cytokines to activate microbiocidal pathways in infected macrophages. However, because these cytokines can also cause tissue damage if over-produced, regulatory immune responses develop, and the balance between pro-inflammatory and regulatory CD4+ T cells responses determines the outcomes of infection. Past studies have identified important roles for pro-inflammatory cytokines such as IFNγ and TNF, as well as regulatory co-inhibitory receptors and the potent anti-inflammatory cytokine IL-10. More recently, other immunoregulatory molecules have been identified that play important roles in CD4+ T cell responses during VL. In this review, we will discuss recent findings about two of these molecules; the NK cell granule protein Nkg7 and the anti-inflammatory cytokine TGFß, and describe how they impact CD4+ T cell functions and immune responses during visceral leishmaniasis.

Vaccine efficacy induced by virus-like particles containing Leishmania donovani surface glycoprotein GP63.

Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice.

Manufacturing and preclinical toxicity of GLP grade gene deleted attenuated Leishmania donovani parasite vaccine.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

Phenotypic and functional characteristics of monocyte subsets in the blood and bone marrow of Indian subjects with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic infection caused by Leishmania donovani in India. L. donovani is an obligate intracellular protozoan residing mostly in macrophages of the reticuloendothelial system throughout chronic infection. Monocytic phagocytes are critical in the pathogenesis of different forms of leishmaniasis. Subsets of monocytes are distinguished by their surface markers into CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes, and CD16++CD14low non-classical monocyte subsets. During cutaneous leishmaniasis (CL), intermediate monocyte are reported to be a source of inflammatory cytokines IL-1ß and TNF, and they express CCR2 attracting them to sites of inflammatory pathology. We examined monocyte subsets in the blood and bone marrow of patients with VL from an endemic site in Bihar, India, and found these contrasted with the roles of monocytes in CL. During VL, intermediate and non-classical CD16+ monocyte subsets expressed instead a non-inflammatory phenotype with low CCR2, high CX3CR1 and low microbicidal oxidant generation, making them more similar to patrolling monocytes than inflammatory cells. Bone marrow CD16+ monocyte subsets expressed a phenotype that might be more similar to the inflammatory subsets of CL, although our inability to obtain bone marrow from healthy donors in the endemic region hampered this interpretation Overall the data suggest that CD16+ intermediate monocyte subsets in VL patients express a phenotypes that contributes to an immunosuppressed pathologic immune state, but in contrast to CL, these do not mediate localized inflammatory responses.

A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection.

Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.

Transcriptomic profiling revealed immune-related signaling pathways in response to experimental infection of Leishmania donovani in two desert lizards from Northwest China.

Little is known about the immune response of lizards to Leishmania parasties. In this study, we conducted the first liver transcriptome analysis of two lizards (Phrynocephalus przewalskii and Eremias multiocellata) challenged with L. donovani, endemic to the steppe desert region of northwestern China. Our results revealed that multiple biological processes and immune-related signaling pathways are closely associated with the immune response to experimental L. donovani infection in the two lizards, and that both lizards show similar changes to mammals in terms of immunity to Leishmania. However, the interspecific divergence of the two lizards leads to different transcriptomic changes. In particular, in contrast to P. przewalskii, the challenged E. mutltiocellata was characterized by the induction of down-regulation of most DEGs. These findings will contribute to the scarce resources on lizard immunity and provide a reference for further research on immune mechanisms in reptiles.

Swarna Bhasma Induces Antigen-Presenting Abilities of Macrophages and Helps Antigen Experienced CD4+ T Cells to Acquire Th1 Phenotypes Against Leishmania donovani Antigens.

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.

Interleukin-7 potentiates MAPK10-elicited host-protective vaccine against Leishmania donovani.

Leishmania donovani causes the potentially fatal disease visceral leishmaniasis for which neither a vaccine nor an adjuvant for human use exists. Although interleukin-7 (IL-7) is implicated in CD4+ T-cell response stabilization, its anti-leishmanial function is uncertain. Therefore, we examined whether IL-7 would potentiate the efficacy of Leishmania major-expressed MAPK10 (LmjMAPK10; M10)-elicited anti-leishmanial host-protective response. We observed that aligning with IL-7R expression, IL-7 increased IFN-γ-secreting TH1 cell but reduced IL-4-producing TH2 cells and production of IL-10 and TGF-ß effectuating anti-leishmanial functions in susceptible BALB/c mouse-derived macrophages. Co-culturing IL-7-pre-treated L. donovani-infected macrophages with L. donovani-infected BALB/c-derived T cells induced IFN-γ-dominated TH1 type anti-leishmanial function. IL-7 treatment of L. donovani-infected BALB/c mice significantly reduced splenic and hepatic parasite loads. Co-culturing CD4+ T cells from IL to 7-treated mice with L. donovani-infected macrophages reduced amastigote numbers suggesting IL-7-elicited host-protective effector T cells. Priming BALB/c with M10 + IL-7 reduced the splenic parasite burden more effectively than that was observed in M10-primed mice. An enhanced protection against L. donovani infection was accompanied by enhanced IL-12 and IFN-γ, but suppressed IL-10 and IL-4, response and host-protective TH1 and memory T cells. These results indicate IL-7-induced leishmanial antigen-specific memory T cell response that protects a susceptible host against L. donovani infection.

Regulatory role of Transcription factor-EB (TFEB) in parasite control through alteration of antigen presentation in visceral leishmaniasis.

Leishmania donovani, an obligate intracellular parasite, the causative agent of visceral leishmaniasis is known to subvert the host immune system for its own survival. Although the precise mechanism is still unknown, emerging evidences indicate that L. donovani efficiently suppress MHC I mediated antigen presentation, rendering inadequate CD8+T cell activation and weakening host defense against parasite. The role of transcription factor EB (TFEB) was recognized in modulating antigen presentation besides its role in lysosomal biogenesis and function. Here, we investigated the regulatory role of TFEB in the modulation of presentation of Leishmania antigen in host tissue. Our results showed an increased expression of TFEB after Leishmania infection both in vitro and in vivo and there was a decrease in the expression of Th-1 cytokine IFNγ along with MHC class I and CD8+T cells indicating attenuation of cell mediated immunity and possibly MHC I restricted antigen presentation. Silencing of TFEB resulted in increased expression of IFNγ and MHC I along with increased CD8+T cells population without any significant change in CD4+T cell number. We also observed a decreased parasite burden in TFEB silenced condition which indicates enhanced parasite clearance by alteration of immunological response possibly through induction of presentation of Leishmania antigen through MHC I. The present study explains the role of TFEB silencing in parasite clearance through regulating the antigen presentation of Leishmania antigen thereby promises to formulate a potential therapeutic strategy against visceral leishmaniasis.

Immunotherapy for visceral leishmaniasis: A trapeze of balancing counteractive forces.

The protozoan parasite Leishmania donovani, residing and replicating within the cells of the monocyte-macrophage (mono-mac) lineage, causes visceral leishmaniasis (VL) in humans. While, Leishmania infantum, is the main causative agent for zoonotic VL, where dogs are the main reservoirs of the disease. The chemotherapy is a serious problem because of restricted repertoire of drugs, drug-resistant parasites, drug-toxicity and the requirement for parenteral administration, which is a problem in resource-starved countries. Moreover, immunocompromised individuals, particularly HIV-1 infected are at higher risk of VL due to impairment in T-helper cell and regulatory cell responses. Furthermore, HIV-VL co-infected patients report poor response to conventional chemotherapy. Recent efforts are therefore directed towards devising both prophylactic and therapeutic immunomodulation. As far as prophylaxis is concerned, although canine vaccines for the disease caused by Leishmania infantum or Leishmania chagasi are available, no vaccine is available for use in humans till date. Therefore, anti-leishmanial immunotherapy triggering or manipulating the host's immune response is gaining momentum during the last two decades. Immunomodulators comprised of small molecules, anti-leishmanial peptides, complex ligands for host receptors, cytokines or their agonists and antibodies have been given trials both in experimental models and in humans. However, the success of immunotherapy in humans remains a far-off target. We, therefore, propose that devising a successful immunotherapy is an act of balancing enhanced beneficial Leishmania-specific responses and deleterious immune activation/hyperinflammation just as the swings in a trapeze.

Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages.

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, provoking liver and spleen tissue destruction that is lethal unless treated. The parasite replicates in macrophages and modulates host microbicidal responses. We have previously reported that neutrophil elastase (NE) is required to sustain L. donovani intracellular growth in macrophages through the induction of interferon beta (IFN-ß). Here, we show that the gene expression of IFN-ß by infected macrophages was reduced by half when TLR4 was blocked by pre-treatment with neutralizing antibodies or in macrophages from tlr2-/- mice, while the levels in macrophages from myd88-/- mice were comparable to those from wild-type C57BL/6 mice. The neutralization of TLR4 in tlr2-/- macrophages completely abolished induction of IFN-ß gene expression upon parasite infection, indicating an additive role for both TLRs. Induction of type I interferon (IFN-I), OASL2, SOD1, and IL10 gene expression by L. donovani was completely abolished in macrophages from NE knock-out mice (ela2-/-) or from protein kinase R (PKR) knock-out mice (pkr-/-), and in C57BL/6 macrophages infected with transgenic L. donovani expressing the inhibitor of serine peptidase 2 (ISP2). Parasite intracellular growth was impaired in pkr-/- macrophages but was fully restored by the addition of exogenous IFN-ß, and parasite burdens were reduced in the spleen of pkr-/- mice at 7 days, as compared to the 129Sv/Ev background mice. Furthermore, parasites were unable to grow in macrophages lacking TLR3, which correlated with lack of IFN-I gene expression. Thus, L. donovani engages innate responses in infected macrophages via TLR2, TLR4, and TLR3, via downstream PKR, to induce the expression of pro-survival genes in the host cell, and guarantee parasite intracellular development.

Genomic and Phenotypic Characterization of Experimentally Selected Resistant Leishmania donovani Reveals a Role for Dynamin-1-Like Protein in the Mechanism of Resistance to a Novel Antileishmanial Compound.

The implementation of prospective drug resistance (DR) studies in the research-and-development (R&D) pipeline is a common practice for many infectious diseases but not for neglected tropical diseases (NTDs). Here, we explored and demonstrated the importance of this approach using as paradigms Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GlaxoSmithKline (GSK) "Leishbox" to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at the genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross-resistance to these drugs, suggesting a new and unique mechanism. By whole-genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at the highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/reduced susceptibility of L. donovani to TCMDC-143345. IMPORTANCE Humans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases but not for NTDs. Here, using Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1-like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.

Immunological characterization of rLdTCP1γ for its prophylactic potential against visceral leishmaniasis in hamster model.

Visceral leishmaniasis (VL) is a chronic tropical disease responsible for devastating epidemics worldwide. Though current treatment relies on drugs, the emergence of resistance, toxic side-effects, and strenuous administration has led to an ineffective remedy. Hence, vaccination remains an alternative and desirable approach for VL control. Though extensive research on anti-leishmanial vaccine candidates has been carried out in past decades, presence of an effective molecule is still missing. In the present study, we have evaluated the immunogenicity and prophylactic potential of a recombinant T-complex protein-1 gamma subunit of L. donovani (rLdTCP1γ), against VL in hamster model. The antigen exhibited in vitro stimulation of lymphoproliferative and NO response in miltefosine and amphotericin B treated hamsters depicting its immunotherapeutic/immunogenic nature. Immunization with rLdTCP1γ revealed a strong protective response against experimental VL as indicated by reduced parasite load in the spleen of immunized group compared to infected control. The immunized animals gained body weight and exhibited significant reduction in the spleen and liver weight as compared to infected controls on days 60, 90, 120 post-challenge. A substantial augmentation of cell-mediated immune response as depicted by an increased lymphocyte proliferation, nitric oxide production, DTH responses and increased levels of IgG2 was observed in rLdTCP1γ immunized hamsters. The Th1 stimulatory potential, imparted by the antigen, was found to be intensified in the presence of adjuvant Bacillus Calmette-Guérin (BCG). The efficacy was further assisted by an upregulated mRNA transcript of Th1 induced cytokines (IL-12, IFN-γ and TNFα) and downregulation of IL-4 and IL-10. The results are thus suggestive of rLdTCP1γ having the potential of a strong vaccine candidate against VL.

Purinergic Enhancement of Anti-Leishmanial Effector Functions of Neutrophil Granulocytes.

Although macrophages are considered for host cells for the multiplication of Leishmania, recent studies indicate the important role of neutrophil granulocytes as host cells for these intracellular parasites. Neutrophils have been shown to be massively and rapidly recruited to the site of Leishmania infection where they represent the first cells to encounter the parasites. Exposure to ATP and UTP have been shown to enhance anti-Leishmania activity of macrophages and intralesional injection of UTP led to strongly reduced parasite load in vivo. Since the in vivo anti-leishmanial effect of extracellular UTP correlated with enhanced neutrophil recruitment and enhanced ROS production at the site of Leishmania infection we hypothesized that exposure to extracellular nucleotides can directly enhance the killing of Leishmania by neutrophils. Since purinergic signaling is an essential mechanism of neutrophil activation the aim of the present study was to assess whether purinergic exposure results in the activation of anti-leishmanial neutrophil functions and, therefore, represent an essential component of enhanced anti-leishmanial defense in leishmaniasis. We could show that exposure to ATP and UTP led to activation and enhanced CD11b expression of primary human neutrophils in vitro. Leishmania-induced ROS production was strongly enhanced by extracellular ATP and UTP. Importantly, exposure to ATP and UTP resulted in enhanced killing of Leishmania donovani by neutrophils. In addition, ATP strongly enhanced the secretion of IL-8 and IL-1ß by Leishmania-exposed neutrophils. Our results suggest that signaling via the P2 receptor and phosphorylation of Erk1/2, Akt and p38 are involved in the purinergic enhancement of anti-leishmanial functions of neutrophils.

Immunomodulatory role of Th17 pathway in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) or Kala-azar is a vector borne protozoan infection caused by Leishmania donovani in the Indian subcontinent mainly India, Nepal and Bangladesh. It is a major public health problem in these countries mostly affecting the socio-economically poor population. Leishmaniasis ranks the third most important disease after malaria and filariasis but is still considered as one of the neglected tropical diseases of the world. For development of better therapeutic agents and effective vaccine against VL, there is a need to understand host immunological changes that play a vital role during course of infection. Therefore, we investigated the role of Th17 pathway in Balb/c mice during Leishmania donovani infection and treatment with amphotericin B. Mice were divided in four groups i.e. Control, Infected, Uninfected treated and Infected treated. The cytokine levels were estimated in the spleen of Balb/c mice on days 1, 3, 7, 14, 17, 21, 28, 35, 45 and 60 post infection and during course of treatment. The mRNA levels of the Th17 pathway during active Leishmania donovani infection and after treatment were determined by real time polymerase chain reaction (RT-PCR) and protein levels by flow cytometry and ELISA. Results of our study revealed that active infection was associated with low levels of Th17 cytokines IL-17, IL-22 and IL-23 and elevated levels of IL-6, IL-1ß and TGF-ß. Amphotericin B treatment restored production of pro-inflammatory cytokines IL-17 and IL-22. The levels of transcription factor RORγt were found to correlate with the levels of IL-17 during infection and also after chemotherapy whereas STAT3 levels were elevated during infection and vice versa after treatment. The findings of this study suggest that Th17 cytokines IL-17 and IL-22 are associated with protection against VL infection and development of any interventions or chemotherapeutic agents targeting Th17 pathway could be an important approach for VL treatment.

Determinants of Innate Immunity in Visceral Leishmaniasis and Their Implication in Vaccine Development.

Leishmaniasis is endemic to the tropical and subtropical regions of the world and is transmitted by the bite of an infected sand fly. The multifaceted interactions between Leishmania, the host innate immune cells, and the adaptive immunity determine the severity of pathogenesis and disease development. Leishmania parasites establish a chronic infection by subversion and attenuation of the microbicidal functions of phagocytic innate immune cells such as neutrophils, macrophages and dendritic cells (DCs). Other innate cells such as inflammatory monocytes, mast cells and NK cells, also contribute to resistance and/or susceptibility to Leishmania infection. In addition to the cytokine/chemokine signals from the innate immune cells, recent studies identified the subtle shifts in the metabolic pathways of the innate cells that activate distinct immune signal cascades. The nexus between metabolic pathways, epigenetic reprogramming and the immune signaling cascades that drive the divergent innate immune responses, remains to be fully understood in Leishmania pathogenesis. Further, development of safe and efficacious vaccines against Leishmaniasis requires a broader understanding of the early interactions between the parasites and innate immune cells. In this review we focus on the current understanding of the specific role of innate immune cells, the metabolomic and epigenetic reprogramming and immune regulation that occurs during visceral leishmaniasis, and the strategies used by the parasite to evade and modulate host immunity. We highlight how such pathways could be exploited in the development of safe and efficacious Leishmania vaccines.

Antibody and cytokine levels in visceral leishmaniasis patients with varied parasitemia before, during, and after treatment in patients admitted to Arba Minch General Hospital, southern Ethiopia.

BACKGROUND: Visceral leishmaniasis is a disease caused by disseminated Leishmania donovani infection which affects almost half a million people annually. Most of the patients are reported from the Indian sub-continent, Eastern Africa and Brazil. In this study, we aimed to determine the levels of antibodies and cytokines in visceral leishmaniasis patients and to examine associations of parasitemia with the clinical states of patients. A prospective study was carried out, enrolling a total of 48 active VL patients who were evaluated before, during different time points and, three months after treatment. Serum cytokine concentrations, antibody levels, parasitemia, laboratory (hematologic and biochemical) measurements, and clinical parameters were assessed. RESULTS: Counts of WBC and platelets, and measurements of hemoglobin (Hb) increased during treatment (P ≤ 0.05). Elevated levels of circulating IL-10, IFN-γ, and TGF-ß1 were measured before treatment. The observed increase in serum IL-10 remarkably declined within 7 days after the start of treatment. Anti-leishmanial antibody index (AI) was high in all VL patients irrespective of spleen aspirate parasite grade before treatment and at different times during treatment. However, a significant (P ≤ 0.05) decrease of AI was observed 120 days post-treatment. IL-2 serum levels were below the detection limit at all sampling points. CONCLUSIONS: The present results suggest that IL-10, IFN-γ, and TGF-ß1 can be used as markers of active visceral leishmaniasis. In addition, measuring circulating cytokines concentrations, particularly IL-10, in combination with other clinical evaluations, could be used as criteria for the cure. The observation that a high serum concentration of IFN-gamma at baseline was associated with low parasitemia deserves further investigations.

Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis.

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.

Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis.

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen-/-) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen-/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen-/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen-/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen-/- immunized hamsters. This study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.